Skin constitutes the largest organ of the human body, serving as a protective barrier against physical injury, radiation and temperature. Skin consists of an underlying mesenchymal (dermal) layer and an outer epithelial (epidermal) layer.
Skin wound healing is regulated by various mechanisms including cell-cell interactions, extracellular matrix production and a number of cytokines and growth factors (see Paul Martin. Wound Healing—Aiming for Perfect Skin Regeneration. Science 276 (75) 1997). Important aims of wound treatment include rapid wound closure and a functionally and aesthetically satisfactory scar.
Wound healing in skin proceeds via an overlapping pattern of events including coagulation, inflammation, tissue formation (epithelialization, formation of granulation tissue, matrix) and tissue remodeling. The process of repair is mediated in large part by interacting molecular signals, primarily cytokines. Initial injury triggers coagulation and an acute local inflammatory response followed by mesenchymal stem cell recruitment, proliferation and matrix synthesis. Failure to resolve the inflammation can lead to chronic non healing wounds, whereas uncontrolled matrix accumulation, can lead to excess scarring.
One of the major growth factors known to be crucial for skin wound healing is fibroblast growth factor-2 (FGF-2), which belongs to a family of similar heparin-binding proteins made by many types of cell, including fibroblasts, melanocytes, endothelial cells and especially stem cells. FGF-2 plays a role in a multitude of developmental and biological processes, including limb development, tissue repair, mesoderm induction, lung development and maintenance of neuron survival; in particular, FGF-2 regulates cell proliferation, migration, and differentiation, including the dermis and epidermis. An FGF-2 variant has been approved by the Food and Drug Administration (FDA) for several Phase-III clinical trials, including peripheral angiogenesis. FGF-2 has both a proliferative and motile effect on keratinocytes, and augments the activities of skin-derived mesenchymal stem cells. FGF-2 is also proto-oncogenic, which makes its use at high concentrations problematic, and alternative therapies are therefore needed.
FGF-2 has a proliferative and motile effect on keratinocytes during wound healing and augments skin-derived mesenchymal stem cells. FGF-2 treatment is effective for the repair of radiation-exposed skin. It's use as a topical treatment for second degree burn wounds has also been evaluated, where significantly faster wound healing as well as larger maximal scar extension, scar retraction to maximal scar extension ratio and elasticity were confirmed. It also supports scarless wound healing by induction of myofibroblast apoptosis but sparing the same effect on fibroblasts.
Re-epithelialization is a critical event in human skin wound healing, in which epidermal keratinocytes laterally migrate to close a wound. In chronic wounds, keratinocyte migration is blocked and the wounds remain open, causing patient morbidity and even fatality. During human skin wound healing, an important step is the initiation of the epidermal and dermal cells at the wound edge to migrate into the wound bed. Human keratinocytes (HKCs) laterally migrate across the wound bed from the cut edge to eventually close the wound (re-epithelialization). The dermal cells, including dermal fibroblasts (DFs) and dermal microvascular endothelial cells (HDMECs), move into the wound following the HKC migration, where these cells deposit matrix proteins, contract and remodel the newly closed wound and build new blood vessels.
Brickman et al. (Glycobiology Vo. 8 No. 5 pp. 463-471, 1998) describe an heparan sulphate called HS2 reported to be capable of interacting with FGF2. HS2 is obtainable from heparan proteoglycans of murine cells at embryonic day 10 as described by Brickman (supra). HS2 has been described as having a molecular weight of approximately 25 kDa and thus, assuming an average molecular mass of 400 Da per disaccharide, consists of about 60 disaccharides. The disaccharide composition of HS2 is set forth in Brickman et al. (Glycobiology Vo. 8 No. 5 pp. 463-471, 1998), WO2010/011185, which is herein incorporated by reference in its entirety. The nitrous acid and heparan lyase digestion profiles of HS2 are shown in FIGS. 29 and 30.
Maccarana et al (Minimal Sequence in Heparin/Heparan Sulfate Required for Binding of Basic Fibroblast Growth Factor. The Journal of Biological Chemistry. Vol. 268, No. 32, Issue 15, pp 23898-23905, 1993) describes experiments investigating the binding of FGF-2 by several small oligosaccharides generated from heparin or HS from human aorta. One octasaccharide fraction (B2) was used to ascribe a structure to the octasaccharide, which the authors called HS-8. It should be noted that this is not the HS-8 of the present invention and the nomenclature is entirely coincidental.
Heparin from pig intestinal mucosa, two samples of selectively O-desulfated heparin, one sample generated by preferential 6-O-desulfation together with N-desulfation of the starting material followed by re-N-sulfation, another sample obtained by selective 2-O-desulfation under alkaline conditions, a low sulphated HS isolated from human aorta, and HS from bovine kidney were used to generate low chain length oligosaccharides of even or odd number.
Even number oligosaccharides were generated from heparin by depolymerisation through partial deaminative cleavage with nitrous acid and the resulting 2,4-anhydro-D-mannose residues were reduced with NAB3H4. Labeled oligosaccharides were separated to generate even numbered species from 4-14 saccharides and a fraction containing 20-24 saccharides. The selectively 6-O-desulfated heparin was similarly treated to yield 4- to 12-saccharides. The isolated and desalted oligosaccharides were subjected to mild acid treatment. Odd numbered heparin oligosaccharides were obtained by further treatment of the 20-24-meric saccharides with heparinase I.
4-14-meric oligosaccharides were generated from human aorta HS by a different strategy involving cleavage at sites occupied by N-acetylated GlcN units. Samples were N-deacetylated and then deaminated with nitrous acid. This treatment leads to deamination of unsubstituted GlcN units and cleavage of glucosaminidic linkages whereas N-sulfated GlcN units remain intact. The products include GlcA-[1-3H]aManR disaccharides (derived from (-GlcNAc)-(GlcA-GlcNac)n- sequences) and GlcA-GlcNSO3-HexA)n-[1-3H]aManR oligosaccharides (derived from (-GlcNac)-GlcA-(GlcNSO3-HexA)n-GlcNac- sequences).
The oligosaccharides generated by these treatments were both short and chemically modified by the process of their preparation, which distinguishes them from the HS of the present invention.